PROJECT SUMMARY Poorly controlled asthma has a substantial economic and medical burden on society. The airways are colo- nized by a complex and diverse airway microbiome that is significantly perturbed in asthma. Enticing past stud- ies suggest that macrolide antibiotics may modify clinical asthma control, particularly in patients with non- eosinophilic, Th2-low asthma. We and others have demonstrated that the central airway microbiome of adult asthmatics is dysbiotic, and that this correlate with worsening airflow obstruction. Whether the airway microbi- ome can be modified by antibiotics such as the macrolide azithromycin, and whether this in turn leads to con- trol of asthma symptoms, is unclear. The major objective of this R34 proposal, a joint collaboration between the asthma investigator teams at the University of Chicago and Northwestern University, is to plan a randomized clinical trial (RCT) that determines whether azithromycin alters the taxonomy, diversity, and metabolic function of the airway microbiome seen in poorly-controlled asthma, and whether this in turn leads to improved clinical control. Our key premise is that understanding how macrolides corrects a dysbiotic airway microbiome may justify the use of macrolides as therapy in poorly controlled, Th2-low asthma. To meet this goal we propose four specific aims. In aim 1 we will conduct pilot trials in patients with poorly controlled, Th2-low asthma to de- termine microbiome responses, treatment effect size and longitudinal microbiome variability. It is critical for us to generate appropriate power calculations, delineate appropriate statistical analyses, and ensure proper study design for a larger RCT. From these short-term pilot studies we will obtain key data that will enable us to plan a successful RCT. In aim 2 we will design a RCT to evaluate the effects of azithromycin on the dysbiotic airway microbiome and on asthma clinical control over 24 weeks in patients with poorly controlled, Th2-low asthma. Working with our biostatistics and informatics cores, we will develop a detailed clinical protocol, determine suit- able statistical analyses for all measures on the microbiome, airway inflammation and clinical control, for the handling of incomplete or missing data, and for any required interim analyses, plan the proper long-term data and biospecimen handling and storage, and develop an appropriate budget for the execution, analysis, and reporting of the proposed RCT. In aim 3 we will satisfy all regulatory requirements and establish all procedures to ensure regulatory compliance for the large clinical trial, including with the FDA and NHLBI. Finally, in aim 4 we will establish operational procedures that will ensure the safe, proper and rigorous execution of the trials. Completion of these aims will prepare our consortium to conduct a comprehensive RCT to determine whether a macrolide antibiotic, by altering the dysbiotic airway microbiome, improves clinical control in poorly controlled Th2-low asthma. Such a trial will provide conclusive evidence of the benefit of this proposed therapy in an asthma endotype that currently has few therapeutic options, demonstrate clearly a proof-of-concept to modu- late inflammatory airways diseases, and will provide substantial benefit to health care.